In vitro genome editing activity of Cas9 in somatic cells after random and transposon-based genomic Cas9 integration

Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2–60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches.


Introduction
Over the years, the domesticated pig has shown to be invaluable as a protein source for human consumption and a diverse animal model for biomedical research. Pigs show a higher anatomical, physiological, and genetic resemblance to humans than rodents, which still represent the Genotyping offspring DNA of the piglets was extracted from tail samples. About 50 mg of tail tissue was lysed in tail lysis buffer (50 mM Tris-HCL, 100 mM NaCl, 100 mM EDTA, 1% SDS, and 40 μl 10 mg/ml proteinase K) overnight at 50˚C, followed by ethanol precipitation. The samples were eluted in aqua bidest and diluted to a concentration of 20 ng/μl for PCR characterization. PCRs for Cas9, neomycin and gRNA integration were performed as described above. The Cas9 amplicon was purified for Sanger sequencing with Invisorb 1 Fragment CleanUp (Invitek Molecular GmbH) diluted to 20 ng/μl and 5 μM of primer was added.
Reverse-transcription qPCR. To determine Cas9 expression in several organs, two boars were sacrificed, one boar with a SB transposon-based integration (SB pig) and one boar with a pX330 random integration (RI pig). Tissue from muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver was homogenized (100mg) and RNA was isolated with TRIzol™ Reagent (Invitrogen) according to manufactures' protocol. Two technical replicates were prepared for RT-qPCR (reverse-transcription qPCR). Isolated RNA was digested with 2 U DNAse I for 30 min at 37˚C prior to cDNA synthesis. Synthesis of cDNA was performed according to the protocol with GoScript™ Reverse Transcriptase (Promega). Quantitative PCR was performed with SYBR™ Green master mix (Life Technologies). Primer sequences for Cas9 expression were the following 5' CCCAAGAGGAACAGCGATAAG 3' and 5' CTATTCTGTGCTGGTGGTGG 3'. Differential mRNA expression was calculated by the Relative Standard Curve Method. Cas9 expression was normalised to the reference gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). A cDNA dilution from pooled muscle RNA was included on every plate to give standard curves for the calculation of relative expression values for Cas9 and GAPDH.

Heritability of vector integration
One cloned and transgenic offspring, boar 762-7 (RI pig), was kept for breeding purposes. After reaching sexual maturity, sperm was collected and frozen according to standard practice. In addition, morphology of the sperm was evaluated. A CASA (Computer Assisted Semen Analysis) analysis was run prior to freezing and after thawing. For in vitro fertilization (IVF), semen was washed with Androhep 1 (Minitube) and centrifuged at 600 x g for 6 minutes. To confirm Cas9 integration in semen, sperm was lysed with tail lysis buffer (see above), 0.5% Trition X 100 (Merck), and 40 mM DTT (1,4-Dithiothreitol, Roth) following DNA ethanol precipitation. Cas9 DNA was amplified as described before. Oocyte collection and maturation was performed as describe above. Different sperm concentrations were evaluated for IVF varying from 100-1500 spermatozoa per oocyte. After fertilization, zygotes were cultured in porcine zygote media (PZM-3). Blastocysts were collected on day six, added to 15 μl cell lysis buffer (described previously), and incubated for one hour at 55˚C, to evaluate Cas9 integration. For artificial insemination, semen was diluted 1:1 in Androhep 1 and transferred twice within 24 hours into a superovulated gilt. The pregnant gilt was sacrificed at day 25 of gestation and fetuses were retrieved from the uterus. DNA was extracted from cephalic parts of the fetuses to detect Cas9 integration.

In vitro activity of Cas9 expression
Fibroblast isolation. Before weaning, fibroblasts were obtained by ear biopsy from transgenic Cas9 positive piglets. Fibroblasts were isolated as previously described [34]. When fibroblasts reached confluency, cells were either frozen or further processed. To obtain Cas9 expressing fetal fibroblasts, boar 762-7 was mated to a wild-type sow, which was slaughtered on day 25 of gestation to retrieve fetuses. Fibroblast cell lines were established from tissues after the removal of excess organs.

Validation of edited cell lines
The edited cells were lysed, and target efficiency and specificity were assessed by flow cytometry and next generation sequencing (NGS).
Next generation sequencing. Knock-out efficiency of gRNA transfected Cas9 expressing fibroblasts was determined by next generation sequencing. The transfected cells were lysed (see above) and B2M, B4GALNT2, and GGTA1 products were amplified by PCR (20 cycles). PCR primers amplifying the target genes are given in S1 Table. Amplicons were purified as described before, and DNA concentrations were determined by the Invitrogen Qubit 4 Fluorometer (ThermoFisher Scientific). DNA of the products was pooled by fragment size to a total concentration of 5 nM and sent for MiSeq sequencing (Illumina). Genome editing efficiency of the generated reads was determined with Geneious Prime Version 2021.0.1. The reads were paired, merged and mapped to the reference gene (NCBI Sus scrofa 11.1).
Flow cytometry. Flow cytometry for B2M and GGTA1 was performed to evaluate the editing efficiency of the integrated Cas9. Flow cytometry to detect expression of B4GALNT2 with Dolichos biflorus agglutinin (DBA) in fibroblasts was unsuccessful.
Lectin -based flow cytometry was performed for GGTA1 edited cells to detect α-galactose expression [38]. In total 0.5 x 10 6 modified and unmodified fibroblast of the same cell line were stained with GSL I-B 4 isolectin conjugated with DyLight 649 (Vector laboratories) for 5 minutes at 37˚C. A previously isolated GGTA1 knock-out cell line [36] severed as negative control.
Off-targets. In total 15 potential off-target regions were amplified. For each gRNA three of the most likely off-target sequences were selected with CRISPOR (http://crispor.tefor.net/) and validated by PCR (S3 Table) and Sanger sequencing. Sequences were aligned to reference sequences (NCBI sus scrofa 11.1).

Generation of Cas9 expressing pigs
Fibroblasts modified with SB transposon-based Cas9 integration were used for SCNT and transferred into six gilts ( Table 2). Out of the six gilts four established pregnancies and delivered 22 SB piglets. Of the 22 piglets 20 were born alive but nine had to be euthanized due to low birth weight and leg deformities related to the SCNT process.
Two gilts were subjected to embryo transfers with fibroblasts modified with random Cas9 integration ( Table 2). In total, 15 RI piglets were born. One was born dead and two of the remaining piglets had to be euthanized due to SCNT related health issues (low birth weight and leg deformities). All others were healthy and developed normally.

Genotyping founder animals
Two SCNT recipients gave birth to 15 piglets (RI pigs) but surprisingly, only two (759-5 and 762-7) carried a Cas9 integration (Fig 2). In contrast, from the transposon integration of which four sows gave birth to 22 piglets (SB pigs) and 15 were positive for Cas9 integration. Purified PCR amplicons were sent for sequencing and aligned to Cas9 sequence (S1 Fig). Reverse-transcription qPCR of organ tissue. Cas9 transcription was confirmed by RT-qPCR in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver. Cas9 expression was normalised to GAPDH and fold changes were calculated. Tissue with random integration of Cas9 showed lower Cas9 expression compared to transposon-based integration, except in liver and tonsils ( Table 3).

Heritability of vector integration
Boar 762-7 (RI pigs) was kept for breeding purposes. After IVF, twelve blastocysts were analyzed to investigate transmission of the Cas9 transgene to the next generation. Ten out of 12 blastocysts revealed a Cas9 integration (Fig 2). One superovulated gilt was artificially inseminated with semen from boar 762-7, (CASA results and Cas9 integration of semen are shown in S2 Fig and S2 Table. On day 25 of gestation, 21 fetuses were retrieved. Genomic analysis revealed Cas9 integration in 11 fetuses (Fig 2).
Flow cytometry B2M. Similar measurements were made for the expression of MHC-I. Cas9 expressing cells transfected with two gRNAs targeting B2M were stained with a swine MHC-I antibody (Fig 4 and Table 6). A B2M knock-out cell line served as negative control Fibroblasts of RI pig 759-5 showed a reduction of 52.44% in MFI and cells from RI pig 762-7 Flow cytometry Cas9 inhibitor. Fetal Cas9 expressing fibroblasts were transfected with the anti-CRISPR AcrIIA4 to inhibit transgenic Cas9 activity and to prove that genome edits resulted from the transgenic Cas9 expression. It was expected that in transgenic Cas9 cells transfected with AcrIIA4 and gRNAs, genome editing of the transgenic Cas9 would be inhibited to a certain extent by AcrIIA4. Fetal fibroblasts from 102-12 were transfected with AcrIIA4and gRNAs (AcrIIA4 inhibitor) or gRNA only (gRNA transfection) Untreated cells served as positive control. Inhibition of Cas9 activity was indicated by a higher MFI for cells treated with gRNA for GGTA1 and AcrIIA4 (64.34) compared to gRNA only treated cells (49.66) ( Table 7 and Fig 5). Cas9 activity was inhibited by AcrIIA4 in B2M gRNA treated cells with an MFI of 26.20 compared to 16.44 MFI in only gRNA treated cells.

Off-targets
Sanger sequencing and alignment to reference sequence after PCR revealed no off-target mutations in the Cas9 expressing cell lines (S3-S7 Figs).

Discussion
Genetically modified pigs pose great opportunities for biomedical research. Due to their close resemblance in physiology and anatomy to humans, pigs are more suitable for human disease modeling compared to rodent models. However, the generation of genetically modified pigs to model diseases human-like require great efforts compared to rodent models. Mainly due to the lack of adequate porcine pluripotent stem cells [41], the production of modified pigs relies on SCNT, microinjection or electroporation of zygotes. Somatic cell nuclear transfer remains inefficient and laborious [42] with only 1-3% success rates [43]. By developing Cas9 expressing breeding lines, germline modifications may be avoided, and the pigs can be used to establish diverse disease models. In this study, we generated Cas9 expressing founder animals based on transposon and random integration. Fibroblasts isolated from the transgenic founder animals were subjected to in vitro gRNA transfection and one RI boar was bred to a wild-type sow. RNA isolated from muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver confirmed Cas9 transcription in organs. As in previously generated Cas9 expressing pigs [21,26], our pigs showed Cas9 genome editing events upon transfection with gRNAs. NGS results revealed editing efficiencies ranging from 0.1 to 2.9% for GGTA1, 3.2 to 27.6% for B2M, and 19.4 to 60.2% for B4GALNT2. It is well established that editing efficiency varies among target loci, as it was also shown in Cre-dependent Cas9 expressing pigs. The study experienced editing efficiencies of 8.1%, 20.2%, and 78.8% for the APC, BRCA1, and BRCA2 loci, respectively, after in vivo transfections of gRNAs [21]. Transposon integration is associated with multicopy integration of transgenes [44][45][46]. Therefore, a multiple Cas9 integration by a SB transposon system was expected to result in higher Cas9 expression. As it was shown in organ tissue, Cas9 expression was overall higher in tissue isolated from the sacrificed SB pig compared to the RI pig. We tried to define copy number and integrations site, to determine copy number differences of transposon-based and random integrations. Nanopore sequencing was performed but with the generated data it was not possible to determine genomic location or copy number of the transgenes. In addition, we investigated potential off-target mutations which could have been induced by the transfected gRNAs. Due to the constant Cas9 expression in the fibroblasts, there is an increased risk for off-target cleavage. Off-targets could have unwanted effects on the genotype of the pigs which could jeopardize the reliability of the disease model. For each gRNA, the three most potential off-target sites in the genome were selected, none of the 15 targets indicated illegitimate cleavage activity. Furthermore, transgenic Cas9 expression may led to activation of the adaptive immune system. Studies which investigated Cas protein as therapeutics have observed specific immune responses towards Cas9 in mice [47][48][49]. However, besides SCNT related health issues our animals grew up healthy. Also, in line with previously generated Cas9 expressing pigs [21,26] the integration and expression of Cas9 had no negative consequences on fertility [21,26]. Similar results were obtained from a Cas9 expressing mouse model to study a variety of diseases and biological functions [20]. In addition, random integration of Cas9 in chickens based on transposon integration [50] or phiC31 integrase [26] did not result in any negative side effects.

Conclusion
In conclusion, we generated functional Cas9 expressing pigs which remained fertile and healthy and therefore are suitable for establishing a Cas9 breeding line. Genome editing of isolated Cas9 expressing fibroblast was feasible, paving the way to generate porcine genotypes for biomedical inquiries. In first investigations on off-target mutations caused by transfected gRNAs were not detected. However, a more detailed investigation of off-target mutations in Cas9 expressing pigs would be necessary when developing human disease models.